The rational development of new antineoplastic agents directed against tubulin, a protein critical for cell division, requires greater understanding of the interaction between the polypeptide subunits of tubulin, its two tightly bound guanine nucleotides, and microtubule-associated proteins. The effects of nucleotides on the stability of microtubules continued to be examined, as were conditions to optimize the separation of alpha-tubulin and beta-tubulin on a preparative scale. The purification of a microtubule-associated protein which causes the formation of microtubule bundles continued to progress and a project to introduce potentially antimitotic nucleotide analogs into cells continued. Polymerization reactions supported by nonhydrolyzable-GTP analogs and ATP were reevaluated. Roles of divalent cations in nucleotide binding to tubulin and in tubulin polymerization were examined. In particular, major differences in effects of Mg 2+ and Be 2+ on tubulin polymerization, tubulin precipitation, polymer stability, and nucleotide binding and hydrolysis were evaluated in detail. A project to define the disulfide bridges in tubulin and in two major classes of microtubule-associated proteins was nearly completed, with the observation that all cysteine sulfhydryl groups in these proteins were free.